近年来,光滑假丝酵母已成为第二位引起侵袭性真菌感染的病原体.光滑假丝酵母对唑类药物(临床一线抗真菌药物)的敏感性低且易发生耐药,一直是研究的热点.介导光滑假丝酵母对唑类药物耐药的关键基因是转录因子pdr1,其功能性突变会使Pdr1蛋白功能过度活跃,导致下游唑类药物外排泵基因高表达,从而对唑类药物耐药.本研究利用同源重组技术,构建在基因pdr1的5′端定点插入3×Flag标签的重组菌株2a2和2b2,为后续利用免疫染色质共沉淀技术寻找Pdr1直接调控基因奠定基础.结果表明,3×Flag标签添加到Pdr1蛋白N端可成功表达Flag-Pdr1蛋白;与野生型菌株相比,表达Flag-Pdr1的菌株对氟康唑的耐药性增强.此外,与野生型菌株相比,表达Flag-Pdr1的菌株中cdr1和pup1基因表达水平显著上升,提示在Pdr1蛋白N端加Flag标签能使其功能活跃,表明N端对Pdr1蛋白功能具有重要意义.%Candida glabrata (C.glabrata) is emerging as the second most common cause for invasive candidiasis and early studies indicate that it is relatively insensitive to azole treatment.Transcription factor Pdr1 is the key regulator of azole resistance genes in C.glabrata.In this study, a Flag sequence was introduced into amino terminus of pdr1 gene at its native locus in chromosome through homologous recombination in clinical isolates of C.glabrata.Western blotting analysis with anti-Flag antibody showed a single immunoreactive species matching the predicted size of Pdr1.Spot assay showed that mutants expressing Flag-Pdr1 were more resistant to fluconazole than wild-type.Real-time quantitative polymerase chain reaction showed that gene expression levels of cdr1 and pup1 in mutants expressing Flag-Pdr1 were significantly increased than those in wild-type strains.
展开▼
